Microparticles labeled with fluorescent dyes have found use in a wide variety of applications. Microparticles are generally considered to be spherical or irregular in shape, and to be less than about 50 micrometers in diameter. They may be prepared by several practical methods from a variety of polymerizable monomers, including styrenes, acrylates and unsaturated chlorides, esters, acetates, amides and alcohols. Microparticles can be further modified by coating with one or more secondary polymers to alter the surface properties of the particles.
Fluorescent microparticles are most commonly used in applications that can benefit from use of monodisperse, chemically inert, biocompatible particles that emit detectable fluorescence and that can bind to a particular substance in the environment. For example, fluorescent particles to which biological molecules have been attached have been used for immunoassays (U.S. Pat. No. 4,808,524 (1989)), for nucleic acid detection and sequencing (Vener, et al. ANALYT. BIOCHEM. 198, 308 (1991); Kremsky, et al., NUCLEIC ACIDS RES. 15, 2891 (1987); Wolf, et al., NUCLEIC ACIDS RES. 15, 2911 (1987)), as labels for cell surface antigens, FLOW CYTOMETRY AND SORTING, ch. 20 (2.sup.nd ed. (1990)), and as tracer to study cellular metabolic processes (J. LEUCOCYTE BIOL. 45, 277 (1989)). The high surface area of microparticles provides an excellent matrix for attaching biological molecules while the fluorescent properties of these particles enable them to be detected with high sensitivity. They can be quantitated by their fluorescence either in aqueous suspension or when captured on membranes.
Fluorescent microparticles can be visualized with a variety of imaging techniques, including ordinary light or fluorescence microscopy and laser scanning confocal microscopy. Three-dimensional imaging resolution techniques in confocal microscopy utilize knowledge of the microscope's point spread function (image of a point source) to place out-of-focus light in its proper perspective. Small, uniform, fluorescently labeled polystyrene microspheres have been employed as point sources for these microscopes (Confocal Microscopy Handbook p. 154 (rev. ed. 1990)).
Many luminescent compounds are known to be suitable for imparting bright and visually attractive colors to various cast or molded plastics such as polystyrene and polymethyl methacrylate. Uniform fluorescent latex microspheres have been described in patents (U.S. Pat. No. 2,994,697, 1961; U.S. Pat. No. 3,096,333, 1963; Brit. U.s. Pat. No. 1,434,743, 1976) and in research literature (Molday, et al., J. CELL BIOL. 64, 75 (1975); Margel, et al., J. CELL SCI. 56, 157 (1982)). A recent patent application of the inventor (Brinkley, et al., Ser. No. 07/629,466, filed 12/18/90) describes derivatives of the dipyrrometheneboron difluoride family of compounds (derivatives of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) as useful dyes for preparing fluorescent microparticles. This family of dyes possesses advantageous spectral data and other properties that result in superior fluorescent microparticles.
Although dipyrrometheneboron difluoride labeled materials are highly fluorescent and photochemically stable, a disadvantage of these fluorescent materials is their relatively small Stokes shift (the difference between the peak excitation and peak emission wavelengths) when only one dye is used. Because the optimum wavelength of the exciting light is close to the peak emission light, fluorescent particles with small Stokes shifts require precise excitation and emission filters to eliminate or reduce interference. The customary use of excitation filters blocks part of the excitation and emission light that would otherwise increase the efficiency of the fluorescence and reduces the intensity of the fluorescent signal. Fluorescent materials that incorporate bright fluorescent dyes with increased Stokes shifts would permit maximum utilization of the available excitation and emission light, resulting in a greater fluorescent signal (see e.g. FIGS. 3A and 3B).
Another advantage of fluorescent materials with large Stokes shifts is that they can be more easily detected in the presence of other fluorescent materials. Immunoassays are typically carried out in body fluids which contain many endogenous fluorescent molecules, such as bilins, flavins and drugs. Since the vast majority of interfering fluorescent materials have relatively short Stokes shifts, the use of a fluorescent label that emits at a wavelength far greater than its excitation wavelength makes the label easier to distinguish from background fluorescence, since its fluorescent signal is emitted at a wavelength at which most background fluorescence is minimal.
A third advantage of fluorescent materials with large Stokes shift is their usefulness in detecting multiple analytes in a single sample using a single excitation wavelength. Using two or more different fluorescent labels, each of which can be excited at a particular wavelength (e.g. the 488 nm argon laser principal emission), the emission peaks of the different labels are detected at different wavelengths, where each emission spectrum is characteristic of a single analyte. In order to successfully accomplish this, the emission peaks of the fluorescent labels must be well-separated from each other so the correction factors between the various dyes are minimized. High photostability of the label is also beneficial. Fluorescent materials with a large Stokes shift can be used in combination with fluorescent materials with a smaller Stokes shift where both materials excite at the same wavelength, but emit at different wavelengths, giving multiple signals that can be resolved using optical filters or monochromators.
Unfortunately, fluorescent compounds useful as labeling reagents that have Stokes shifts of 50-100 nm as well as high fluorescence efficiency and emission wavelengths of greater than 500 nm required for detectability are relatively rare. (Haugland, Fluorescein Substitutes for Microscopy and Imaging, OPTICAL MICROSCOPY FOR BIOLOGY pp. 143-57 (1990). The magnitude of the Stokes shift in fluorescent dyes has been found to be generally inversely proportional to the high absorbance needed to ensure a strong signal. Fluorescent dyes in use as labeling reagents for biological molecules, such as xanthenes, dipyrrometheneboron difluorides, rhodamines and carbocyanines commonly have Stokes shifts of less than about 30 nm.
The lack of suitable fluorescent dyes with large Stokes shifts has led to the development and use of protein-based fluorophores known as phycobiliproteins as labels (e.g. U.S. Pat. Nos. 4,520,110 and 4,542,104 both to Stryer, et al. (1985)). Like other fluorophores, they have been covalently attached to beads and macromolecules. See, e.g., Oi, et al., J. CELL BIO. 93,981 (1982). These large bilin-containing molecules have the desirable characteristics of very high extinction coefficients and they use internal energy transfer between unlike, covalently-linked fluorophores to accomplish a relatively large Stokes shift. They have the disadvantage of poor chemical stability, instability to photobleaching, limited long wavelength emission capability, bulky molecular size (MW &gt;100,000 Daltons) and relatively high cost. Furthermore, only a few proteins of this type are known and one cannot select or appreciably adjust their spectral properties. In an effort to improve the fluorescent emission efficiency of phycobiliproteins without significantly increasing their molecular size, phycobiliproteins have been covalently coupled to the fluorescent dye Azure A (U.S. Pat. No. 4,666,862 to Chan (1987)).
It is known that covalent coupling of a pair of fluorophores results in a fluorescent dye with a larger Stokes shift than either of the individual dyes (e.g. Gorelenko, et al., Photonics of Bichromophores Based on Laser Dyes in Solutions and Polymers, EXPERIMENTELLE TECHNIK DER PHYSIK 37, 343 (1989)). This approach, although reportedly effective in increasing the Stokes shift, requires complex synthetic procedures to chemically couple the two dyes together and are limited by the number and location of available reactive sites. The process of carrying out the necessary synthetic procedures to attach three, four, or more dyes sufficiently close together and in the proper configuration to undergo substantial energy transfer would be exceedingly difficult, if not impossible. Furthermore, covalently linked molecules typically have sufficient freedom of movement that significant collisional deactivation occurs, leading to loss of energy by vibrational relaxation rather than by fluorescence. There is a need for a way of combining the spectral properties of dyes by methods other than complex covalent coupling to provide useful fluorescent labels with an enhanced effective Stokes shift.
In studies of energy transfer between pairs of covalently linked dyes, it has been shown that the efficiency of energy transfer between two fluorescent dyes is inversely proportional to the sixth power of the distance between the two interacting molecules, consistent with Forster's theory (Stryer & Haugland, Energy Transfer: A Spectroscopic Ruler, PROC. NAT'L ACAD. SCI. USA 58, 719 (1967)). The reference suggests that the percentage of measurable energy transfer can be used to measure the distance separating the covalently linked fluorophores in the 10 to 60 .ANG. range. A subsequent paper, Haugland, Yguerabide, & Stryer, Dependence of the Kinetics of Singlet-Singlet Energy Transfer on Spectral Overlap, PNAS 63, 23 (1969), reported that intramolecular singlet energy transfer depends on the magnitude of spectral overlap integral.
Energy transfer has been demonstrated between dyes that have been coupled to macromolecules to study intramolecular distances and conformation in biomolecules, e.g., Julien & Garel, BIOCHEM. 22, 3829 (1983); Wooley, et al., BIOPHYS. CHEM. 26, 367 (1987); and in polymer chains and networks, e.g. Ohmine, et al., MACROMOLECULES 10, 862 (1977); Drake, et al., SCIENCE 251, 1574 (1991). Energy transfer with resultant wavelength shifting has also been described for mixtures of dyes in lasing solutions, e.g. Saito, et al., APPL. PHYS. LETT. 56, 811 (1990). Energy transfer has been demonstrated between monomolecular layers of dyes and other organized molecular assemblies, e.g. Kuhn, Production of Simple Organized Systems of Molecules, PURE APPL. CHEM. 11, 345 (1966), abstracted in CHEM. ABSTRACTS 66, 671 (1967); Yamazaki, et al., J. PHYS. CHEM. 94, 516 (1990). Energy transfer between paired donor and acceptor dyes has also been demonstrated in polymer films as a way of studying the energy transfer dynamics, e.g. Mataga, et al., J. PHYS. CHEM. 73, 370 (1969); Bennett, J. CHEM. PHYSICS 41, 3037 (1964). Although the conformity of research results to Forster's theoretical formulation have been widely reported, utilitarian applications of the theory have been limited. The cited references neither anticipate nor suggest fluorescent microparticles incorporating a series of dyes to be used as labeling reagents with an enhanced effective Stokes shift.
It is an object of the invention to provide a more simple method than complex covalent coupling for combining the spectral properties of multiple dyes, while minimizing collisional deactivation, allowing efficient energy transfer and increasing the effective Stokes shift for the purpose of providing more useful fluorescent labeling reagents. It is a further object of the invention to provide materials that have not only an increased effective Stokes shift but materials for which the effective Stokes shift can be selectively controlled by the selection of appropriate dyes with overlapping spectral properties.
Immobilizing fluorescent dyes randomly in a polymeric matrix according to the subject invention provides just such a simple method of providing novel fluorescent materials with controllable, enhanced effective Stokes shifts. Certain fluorescent dyes, such as dipyrrometheneboron difluoride dyes, coumarin dyes and polyolefin dyes, have high fluorescence efficiency when incorporated into polymeric materials and are available in a large number of derivatives with a wide range of excitation and emission maxima, These characteristics allow the wavelength of excitation and the magnitude of the increase in the effective Stokes shift to be easily controlled by carefully selecting dyes with the appropriate spectral overlap for incorporation into the microparticles.